Blood vessels are required for organ formation and homeostasis during embryonic development and throughout adult life. Since the signaling mechanisms that govern blood vessel formation are largely conserved and are similar during embryonic and adult stages, it is possible to utilize model organisms to gain insight into this process. Blood vessel formation occurs through the coordination of a complex array of cellular behaviors. Recent evidence indicates that the Notch signaling pathway plays an essential role during this process to determine cell fates and modulate signaling output during vascular development. Notably, Notch is essential for promoting arterial endothelial cell fate while during angiogenesis it helps to limit an endothelial cell's response to pro-angiogenic cues. However, little is known about when and where Notch activation occurs during the early stages of vascular development or how Notch activation affects endothelial cell fates at different steps of blood vessel formation. Furthermore, few Notch target genes have been identified that may mediate the effects of Notch activation in endothelial cells. In the studies proposed here, we will take advantage of the zebrafish as a model system to address the dynamic role of Notch signaling during blood vessel development. Using a transgenic indicator line that is transcriptionally responsive to Notch signaling, we will visualize the temporal and spatial dynamics of Notch activation in developing blood vessels through time-lapse analysis in live zebrafish embryos. In addition, we will establish transgenic lines expressing a photoconvertible fluorescent protein in all endothelial cells or in Notch-positive endothelial cells. These lines will allow detailed fate mapping of Notch-positive and Notch-negative endothelial cells during multiple stages of vascular development. We will also characterize the function of putative direct targets of Notch in endothelial cells by taking advantage of our ability to generate zebrafish knockout lines using zinc finger nucleases. This will enable the rapid generation of null alleles in target genes of interest to determine their role downstream of Notch. Importantly, this technique will also allow us to delete endogenous Notch responsive cis elements in genes of interest to definitively characterize the role of Notch in regulating downstream targets. Finally, we will combine global expression profiling and genome-wide assay of occupancy at Notch binding sites to characterize the Notch-responsive transcriptional network in endothelial cells.
A crucial step in the formation of new blood vessels is the establishment of proper blood vessel identity (e.g. artery versus vein). Recent evidence suggests that loss of blood vessel identity in a number of congenital human diseases can lead to vascular malformations. The studies in this proposal will address how blood vessel identities are determined during development.
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